Erratum in

Abstract

Fission yeast is a powerful model organism that has provided insights into important cellular processes thanks to the ease of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in Schizosaccharomyces pombe. In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe.

The fluoride channels from S. pombe (Fex1p and Fex2p) and S. cerevisiae (FEX1 and FEX2) share only 36% identical amino acids but have several conserved regions that contain amino acids with similar properties (boxes). See also . The alignment was performed with Clustal Omega. Symbols: asterisk (*), fully conserved residues; colon (:), residues with strong similar properties; point (.), residues with weak similar properties

Spot assays showing that growth of wild-type strains on solid media is insensitive to fluoride up to 5 mm, and growth of the fex1Δ fex2Δ double mutant is impaired starting at 10 μm but can be rescued by expression of Fex1p from a plasmid (also expressing Cas9)

Growth of S. pombe in the presence of different concentrations of fluoride. (A) Growth of the wild-type strain in liquid media is insensitive to fluoride concentrations up to 5 mm. (B) Growth of the fex1Δfex2Δ strain is similar to wild-type in the absence of fluoride but its growth is impaired for fluoride concentration higher than 10 μm. (C) Growth of the fex1Δfex2Δ strain on fluoride can be rescued by the expression of Fex1p on a plasmid. Note that the time-scale for this figure is longer than for other figures because the expression of Cas9 from the plasmid delays the growth of S. pombe. (D) The absence of expression of Cas9 allows the growth of S. pombe to be rescued with growth curves similar to wild-type

Comparison of timelines using auxotrophic or fluoride channel markers, as observed in genome edition using CRISPR/Cas9. (Top) Using a Fex1p-expressing plasmid and selection on rich media with fluoride allows the screening for positive colonies 5 days after the initial transformation. (Bottom) Using a ura4+-expressing plasmid and minimal media lacking uracil allows the screening for positive colonies after at least 11 days